Treatment of skin conditions with oxysterol activators of LXRα

ABSTRACT

Disorders of the skin and mucous membranes that have a disrupted or dysfunctional epidermal barrier are treated or prevented by topical application of compounds that are either activators of the farnesoid X receptor, activators of the peroxisome proliferator-activated receptor alpha, and oxysterol activators of the LXRalpha receptor. The same compounds are also effective in treating disorders of epidermal differentiation and proliferation.

CROSS REFERENCE TO RELATED APPLICATION

This application is a division of application Ser. No. 09/101,366, filedJun. 16, 1999, which is the national phase of PCT/US98/01276 filed Jan.22, 1998, and a continuation-in-part of U.S. priority application Ser.No. 08/788,973, filed Jan. 24, 1997 abandoned, all such application areincorporated herein by reference.

GOVERNMENT RIGHTS

This invention was made at least in part with assistance from the UnitedStates Federal Government, under Grant No. HD 29706 of the NationalInstitutes of Health. As a result, the Government has certain rights inthis invention.

This invention resides in the technical field of topical formulationsfor application to skin, and to the treatment of subjects suffering fromskin or mucous membrane diseases or disorders which display disruptionsof the barrier function, and those that involve disorders of epidermaldifferentiation and proliferation.

BACKGROUND OF THE INVENTION

One of the functions served by the epidermis in mammals is to form abarrier against excessive transcutaneous water loss to the environment.This barrier is formed by the anucleate, cornified, outermost layers ofthe epidermis, collectively known as the stratum corneum. Localized orgeneralized perturbations of the epidermal barrier occur in a variety ofdiseases and conditions of the skin and mucous membrane. Theseperturbations not only contribute significantly to the morphology ofcutaneous lesions, but also activate certain skin diseases such as theKoebner phenomenon in psoriasis and the inflammation in eczematousdisorders. The integrity of the barrier is also known to be a majorfactor in regulating epidermal DNA synthesis. Thus, maintenance of anormal epidermal barrier is a physiological means of inhibitingepidermal hyperproliferation. Examples of conditions that involve orgive rise to a disrupted or dysfunctional epidermal barrier are:

fluid and electrolyte abnormalities, hypothermia, and infection throughthe skin in premature infants less than 33 weeks of gestational age;

inflammation to mucous membranes, such as cheilitis, chapped lips, nasalirritation and vulvovaginitis;

eczematous dermnatitides, such as atopic and seborrheic dermatitis,allergic or irritant contact dermatitis, eczema craquelée, photoallergicdermatitis, phototoxic dermatitis, phytophotodermatitis, radiationdermatitis, and stasis dermatitis;

ulcers and erosions resulting from trauma, burns, bullous disorders, orischemia of the skin or mucous membranes;

several forms of ichthyoses;

epidermolysis bullosae;

psoriasis;

hypertrophic scars and keloids;

cutaneous changes of intrinsic aging and photoaging;

frictional blistering caused by mechanical shearing of the skin; and

cutaneous atrophy resulting from the topical use of corticosteroids.

The key constituents of the epidermis that are needed for a functionalbarrier are the intercellular, lamellar bilayer sheets of stratumcorneum lipids. The synthesis of stratum corneum lipids is relativelyautonomous from circulating or dietary influences. The syntheticresponse is regulated instead by alterations in permeability barrierfunctions. The regulation occurs through changes in the activities,phosphorylation (activation) state, mass, and mRNA for the rate-limitingenzymes of each of the three key lipids: serine palmitoyl transferase(for ceramides), HMGCoA reductase (for cholesterol), and both acetyl CoAcarboxylase and fatty acid synthase (for fatty acids). Other results ofalterations in barrier function are the regulation of key enzymes ofextracellular lipid processing. One such enzyme is β-glucocerebrosidase,which catalyzes the conversion of precursor glycosylceramides intoceramides.

While permeability barrier requirements regulate lipid synthesis, theendogenous regulators of barrier development and homeostasis are notknown. Recent studies from the inventors' laboratories have shown thatseveral activators and ligands of the nuclear receptor superfamily, suchas glucocorticoids, thyroid hormone, and estrogen, accelerate theappearance of a mature barrier in fetal rodent skin. Hanley, K., et al.,“Epidermal barrier ontogenesis: maturation in serum-free media andacceleration by glucocorticoids and thyroid hormone but not selectedgrowth factors,” J. Invest. Derrnatol. 106:404-411 (1996); Hanley, K.,et at., “Hormonal basis for the gender difference in epidermal barrierformation in the fetal rat. Acceleration by estrogen and delay byandrogen,” J. Invest. Dennatol. 97:2576-2584 (1996). In contrast, othermembers of this family, such as 1,25-dihydroxy vitamin D₃ 9-cis-retinoicacid, and all-trans-retinoic acid, had no effect.

SUMMARY OF THE INVENTION

It has now been discovered that the formation of a mature, fullydifferentiated stratum corneum and a functional epidermal permeabilitybarrier are accelerated by the topical administration of certainactivators of any one of three nuclear receptors—the farnesoidX-activated receptor (FXR), the peroxisome proliferator-activatedreceptor α (PPARα), and the liver-based receptor known as LXRα. Thesethree receptors are nuclear receptors and are part of the nuclearreceptor superfamily of transcription factors. The three receptorsreside in a subgroup of the superfamily, all receptors in the subgroupsharing the feature that they function only when having formedheterodimers with the retinoid X receptor (RXR). Many other members ofthe subgroup however do not have activators that accelerate theformation of a mature stratum corneum or barrier development—theseinclude the vitamin D receptor (VDR), the all-trans-retinoic acidreceptors (RARα,β,δ), and the 9-cis-retinoic acid (RXR) receptor. Theability of FXR, PPARα and LXRα activators to achieve this result istherefore unique among members of this subgroup.

The ability of FXR activators to accelerate barrier development isparticularly surprising since compounds similar in structure to farnesol(a prominent FXR activator) that are not themselves FXR activators donot accelerate barrier development, despite the similarity in structureto those that do. Also surprising is the ability of the PPARα receptor,since other PPAR receptors exist (with their own separate activators)that are similar in structure and function, and yet only activators ofthe PPARα receptor accelerate barrier development. A further surprisingaspect of this discovery is that the barrier development accelerationassociated with PPARα activation is not related to any distinctionbetween essential and non-essential fatty acids, but rather to certaincommon structural features. A still further surprising aspect of thediscovery relates to oxysterols that are not activators of LXRα but arevery close in structure to those that are. The oxysterols that are notLXRα activators do not produce the beneficial results of this inventiondespite their similarity in structure. Furthermore, many of theactivators that are the subject of this invention have never before beenknown to have any utility as topical epidermal agents.

This newly discovered activity of the three classes of activatorsrenders them useful in the treatment of mammalian skin sufferingdeficient or perturbed barrier function. The invention is particularlyuseful in the treatment of premature infants, particularly those lessthan 33 weeks of gestational age. This invention is also useful foralterations in epidermal differentiation and proliferation. Applicationsinclude melanoma and non-melanoma skin cancers and skin precancers,disorders of epidermal differentiation and proliferation such aspsoriasis, atopic dermatitis, and various types of ichthyosis with orwithout an associated barrier abnormality; and benign neoplasms such aswarts, condylomata, and seborrheic keratoses.

Other features and advantages of the invention will become apparent fromthe description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing trans-epidermal water loss (TEWL) testresults of six compounds, two of which are within the scope of thisinvention.

FIG. 2 is a bar graph showing TEWL test results of five additionalcompounds, three of which are within the scope of this invention.

FIG. 3 is a bar graph showing TEWL test results of seven additionalcompounds, one of which is within the scope of this invention.

FIG. 4a is a plot of TEWL vs. concentration of active ingredient for twocompounds within the scope of this invention.

FIG. 4b is a plot of TEWL vs. concentration of active ingredient for athird compound within the scope of this invention.

FIG. 4c is a plot of TEWL vs. concentration of active ingredient for afourth compound and a fifth compound within the scope of this invention.

FIG. 5a is a bar graph showing TEWL test results for two compoundswithin the scope of this invention at suboptimal levels; bothindividually and in combination.

FIG. 5b is a bar graph showing TEWL test results for the same twocompounds as FIG. 5a except at optimal levels, both individually and incombination.

FIG. 6a is a bar graph showing levels of involucrin mRNA in cellcultures treated with clofibrate.

FIG. 6b is a bar graph showing levels of transglutaminase mRNA in cellcultures treated with clofibrate.

FIG. 7a is a further bar graph showing levels of involucrin mRNA in cellcultures treated with clofibrate.

FIG. 7b is a further bar graph showing levels of transglutaminase mRNAin cell cultures treated with clofibrate. The calcium ion levels in themedia represented by FIGS. 7a and 7 b are higher than those of FIGS. 6aand 6 b.

FIG. 8 is a bar graph showing levels of involucrin and transglutaminasemRNA in cell cultures treated with clofibrate at different doses.

FIG. 9a is a bar graph showing levels of involucrin protein in cellcultures treated with clofibrate at different doses.

Further FIG. 9b is a further bar graph showing similar data derived frommedia containing a higher calcium content.

FIG. 10 is a bar graph showing levels of involucrin protein in cellcultures treated with farnesol at different doses.

FIG. 11 is a bar graph showing levels of involucrin protein in cellcultures treated with juvenile hormone III at different doses.

FIG. 12 is a bar graph showing levels of increase in the rate ofcornified envelope formation in cell cultures treated with farnesol andjuvenile hormone III at different doses.

FIG. 13 is a bar graph showing the degree of decrease in DNA content incell cultures treated with farnesol and juvenile hormone III atdifferent doses.

FIG. 14a is a bar graph showing levels of involucrin andtransglutaminase mRNA levels in cell cultures treated with two LXRαactivators as well as two related compounds (for comparison).

FIG. 14b is a bar graph with similar data and a comparison against afurther related compound.

FIG. 15 is a bar graph showing the dependency of involucrin andtransglutaminase mRNA levels on the dosage of an LXRα activator.

FIG. 16 is a bar graph showing results similar to those of FIGS. 14a and14 b except using a cell culture with a higher calcium content.

FIG. 17a is a bar graph showing involucrin and transglutaminase proteinlevels in cell cultures treated with two LXRα activators as well as onetreated with cholesterol for comparison.

FIG. 17b is a bar graph showing the dependency of the transglutaminaseprotein level on the dosage of one of the LXRα activators at highcalcium concentration.

FIG. 17c is a bar graph showing the involucrin protein level in cellcultures treated with two LXRα activators and one with cholesterol athigh calcium concentration.

FIG. 18a is a bar graph showing cornified envelope formation inlow-calcium cell cultures treated with two LXRα activators as well asone treated with cholesterol for comparison.

FIG. 18b is a bar graph showing similar results in high-calcium cellcultures.

FIG. 19 is a bar graph showing the rate of DNA synthesis in low-calciumcell cultures treated with two LXRα activators as well as one treatedwith cholesterol for comparison.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The farnesoid X-activated receptor (FXR), the peroxisomeproliferator-activated receptor a (PPARα), and the receptor LXRα aremembers of a superfamily of approximately 150 proteins that bind tocis-acting elements in the promoters of their target genes and modulategene expression in response to hormone activators or ligands. For manyof these receptors, the activators are known, while for others, termed“orphan receptors,” the activators are unknown. Furthermore, some ofthese receptors bind to their target genes as dimers consisting of twomolecules of the same receptor (homodimers), while others bind as dimersconsisting of one molecule each of two different receptors(heterodimers). Prominent among the latter are nuclear receptors thatrequire heterodimerization with the retinoid X receptor, as disclosed byYu, V. C., et al., “RXRβ: a coregulator that enhances binding ofretinoic acid, thyroid hormone, and vitamin D receptors to their cognateresponse elements,” Cell 67:1251-1266 (1991). Members of this groupinclude the vitamin D receptor, the thyroid hormone receptor (T₃R), theretinoic acid receptor (RAR), the farnesoid X-activated receptor (FXR),the peroxisome proliferator-activated receptors (PPAR), and LXRα.

The farnesoid X-activated receptor (FXR) was first reported by Formanand coworkers, Forman, B. B., “Identification of a nuclear receptor thatis activated by farnesol metabolites,” Cell 81:687-693 (1995). Thisreceptor is a protein having a relative molecular mass (M_(r)) ofapproximately 54,000, and is a vertebrate transcription factor regulatedby intracellular metabolites. The receptor is activated by certainfarnesoids, i.e., farnesol itself and compounds derived from, and/orsimilar in structure to, farnesol. These farnesoids include farnesol,farnesal, farnesyl acetate, farnesoic acid, geranylgeraniol, andjuvenile hormone III. The chemical name for farnesol is3,7,11,trimethyl-2,6,10-dodecatrienol, and the chemical name forjuvenile hormone III is7-methyl-9-(3,3-dimethyloxiranyl)-3-methyl-2,6-nonadienoic acid methylester. Farnesoids and metabolites that do not activate the FXR aregeraniol, squalene, methoprene, mevalonate, squalene oxide, squalenedioxide, lanosterol, 24,25-epoxycholesterol, pregnenalone,dehydroepiandrosterone, bile acids, and 25-hydroxycholesterol. FXRactivators of particular interest are farnesol (denotingtrans,trans-farnesol hereinafter), farnesal, methyl farnesyl ether,ethyl farnesyl ether, methyl farnesoate, ethyl farnesoate,7-methyl-9-(3,3-dimethyloxiranyl)-3-methyl-2,6-nonadienoic acid methylester, and 7-methyl-9-(3,3-dimethyloxiranyl)-3-methyl-2,6-nonadienoicacid ethyl ester. Preferred among these are farnesol, farnesal, methylfarnesyl ether, methyl farnesoate, and7-methyl-9-(3,3-dimethyloxiranyl)-3-methyl-2,6-nonadienoic acid methylester. Particularly preferred are farnesol and7-methyl-9-(3,3-dimethyloxiranyl)-3-methyl-2,6-nonadienoic acid methylester juvenile hormone III).

Peroxisome proliferator-activated receptors (PPAR) are described in areview article by Schoonjans, K., “Role of the peroxisomeproliferator-activated receptor (PPAR) in mediating the effects offibrates and fatty acids on gene expression,” J. Lipid Res. 37:907-925(1996). Three subtypes of PPAR have been identified, and these aredesignated as α, β (or δ), and γ. The α subtype has been cloned fromXenopus, humans, mouse and rat; the β (or δ) subtype from Xenopus,humans and mouse; and the γ subtype from Xenopus, humans and hamster.The PPARs have a modular structure consisting of six functional domains.The one domain that serves as the DNA-binding domain contains about 66amino acids and is stabilized by two zinc atoms, each binding to fourinvariant cysteine residues. Included among the activators for PPARα arefibrates, and fatty acids other than short-chain (<C₁₀) fatty acids,long-chain monounsaturated fatty acids, and dicarboxylic acids,particularly dodecanedioic acid. Also included are lower alkyl,preferably methyl, esters of the fibrates and lower alkyl, preferablymethyl, esters of the fatty acids. Fibrates include:

clofibrate: 2-(4-chlorophenoxy)-2-methylpropanoic acid ethyl ester

fenofibrate: 2-(4-(4-chlorobenzoyl)phenoxy)-2-methylpropanoic acidisopropyl ester

ciprofibrate: 2-(4-(2,2-dichlorocyclopropyl)phenoxy)isobutyric acid

gemfibrozil: 2-(2,4-dimethylphenoxypropyl)-2-methylpropanoic acid

bezafibrate: 2-(4-(4-chlorobenzoylaminoethyl)phenoxy)-2-methylpropanoicacid

Among the fatty acids, substituted fatty acids are particularly potentactivators. PPARα activators of particular interest are linoleic acid,oleic acid, 5,8,11,14-eicosatetraynoic acid,(4-chloro-6-(2,3-xylidino)-2-pyrimidinyl)thioacetic acid, andclofibrate. A list including these and other examples of PPARαactivators is as follows:

2,4-dichlorophenoxyacetic acid

2,4,5-trichlorophenoxyacetic acid

2-methyl-4-chlorophenoxyacetic acid

2-phenoxy-2-methylpropanoic acid ethyl ester

2-(4-bromophenoxy)-2-methylpropanoic acid ethyl ester

2-(4-iodophenoxy)-2-methylpropanoic acid ethyl ester

2-(2-chlorophenoxy)-2-methylpropanoic acid ethyl ester

2-(3-chlorophenoxy)-2-methylpropanoic acid ethyl ester

2-(4-chlorophenoxy)-2-methylpropanoic acid ethyl ester

2-(4-(4-chlorophenyl)phenoxy)-2-methylpropanoic acid ethyl ester

2-(4-(4-chlorobenzoyl)phenoxy)-2-methylpropanoic acid isopropyl ester

2-(4-(2,2-dichlorocyclopropyl)phenoxy)-2-methylpropanoic acid

2-(4-(4-chlorobenzoylaminoethyl)phenoxy)-2-methylpropanoic acid

2-(2, 3-dimethyl-4-(1,2,3 ,4-tetrahydronaphth-1 -yl)phenoxy)acetic acid

2-(2-methyl-3-ethyl-4-(4-chlorobenzyl)phenoxy)acetic acid

(4-chloro-6-(2,3-xylidino)-2-pyrimidinyl)thioacetic acid

2-((4-chloro-6-(2,3-xylidino)-2-pyrimidinyl)thioacetamido)ethanolperfluoro-n-decanoic acid

di-(2-ethylhexyl)adipate

di-(2-ethylhexyl)phosphate

di-(2-ethylhexyl)sebacate

bis-(carboxymethylthio)-1,10-decane

ethyl 4-(4-chlorophenoxy)butanoate

2-(2-nitro-5-(2-chloro-4-trifluoromethylphenoxy)benzoyloxy)propanoicacid ethyl ester

2-(4-(4-chlorobenzoyl))phenoxy-2-(2-methylpropionamido)ethylsulfonicacid

tetradecyloxyacetic acid

tetradecyloxypropionic acid

perfluorobutanoic acid

perfluorooctanoic acid

tetradecylthioacetic acid

tetradecylthiopropionic acid

di-(2-ethylhexyl)phthalate

mono-(2-ethylhexyl)phthalate

2-ethylhexanoic acid

2-propylhexanoic acid

The receptor LXRα was first described by Willy, P. J., et al., “LXR, anuclear receptor that defines a distinct retinoid response pathway,”Genes & Development 9:1033-1045 (Cold Spring Harbor Laboratory Press),and is named LXRα due to its initial isolation from the liver and itsliver-rich expression pattern. The activators of LXRα are a subset ofoxysterols, including 7α-hydroxycholesterol, 25-hydroxycholesterol,27-hydroxy-cholesterol, 4β-hydroxycholesterol, 24-hydroxycholesterol,20(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and20,22-dihydroxycholesterol. Structurally similar compounds that are notactivators of LXRα include cholesterol itself and the oxysterols7,25-dihydroxycholesterol, 17α-hydroxycholesterol, and22(S)-hydroxycholesterol (enantiomer of 22(R)-hydroxycholesterol). Thenumbering convention used for substituted holesterols is as follows:

The term “activator” is used in this specification to denote anymolecular species that results in activation of the indicated receptor,regardless of whether the species itself binds to the receptor or ametabolite of the species binds to the receptor when the species isadministered topically. Thus, the activator can be a ligand of thereceptor or it can be an activator that is metabolized to the ligand ofthe receptor, i.e., a metabolite that is formed in tissue and is theactual ligand.

In the practice of this invention, the activators will be administeredas active ingredients in a formulation that is pharmaceuticallyacceptable for topical administration. These formulations may or may notcontain a vehicle, although the use of a vehicle is preferred. Preferredvehicles are non-lipid vehicles, particularly a water-miscible liquid ormixture of liquids. Examples are methanol, ethanol, isopropanol,ethylene glycol, propylene glycol, and butylene glycol, and mixtures oftwo or more of these compounds.

The concentration of active ingredient in the vehicle will generallyrange from about 10 μM to about 1000 μM, although for certain activeingredients, the concentration may vary outside this range. Informulations containing farnesol as the active ingredient, preferredconcentrations are in the range of about 10 μM to about 100 μM. Informulations containing juvenile hormone III as the active ingredient,preferred concentrations are in the range of about 10 μM to about 200μM. In formulations containing clofibrate as the active ingredient,preferred concentrations are in the range of about 100 μM to about 1,000μM. In formulations containing oleic acid as the active ingredient,preferred concentrations are in the range of about 100 μM to about 1000μM. In formulations containing linoleic acid as the active ingredient,preferred concentrations are in the range of about 5 μM to about 50 μM.

Topical formulations containing the FXR or PPARα activators inaccordance with the present invention are applied to beneficial effectto skin and/or mucus membranes. The activators can be formulated aslotions, solutions, gels, creams, emollient creams, unguents, sprays, orany other form that will permit topical application. The formulation mayalso contain one or more agents that promote the spreading of theformulation over the affected area, but are otherwise biologicallyinactive. Examples of these agents are surfactants, humectants, wettingagents, emulsifiers, or propellants.

Amounts that are referred to herein as effective in enhancing barrierdevelopment are any amount that will cause a substantial relief of thesymptoms of a disrupted or dysfunctional epidermal permeability barrierwhen applied repeatedly over time. The optimum amounts in any giveninstance will be readily apparent to those skilled in the art or arecapable of determination by routine experimentation.

Examples of skin conditions that are susceptible to treatment by thepractice of this invention are:

the skin of premature infants of gestational age less than 33 weeks;

atopic and seborrheic dermatitis;

inflammation to mucous membranes, such as cheilitis, chapped lips, nasalirritation and vulvovaginitis;

eczematous dermatitis resulting from allergic and irritant contact,eczema craquelee, radiation and stasis dermatitis;

ulcers and erosions due to chemical or thermal burns, bullous disorders,or vascular compromise or ischemia including venous, arterial, embolicor diabetic ulcers;

ichthyoses, with or without an associated barrier abnormality;

epidermolysis bullosa;

psoriasis;

hypertrophic scars and keloids;

intrinsic aging and/or dermatoheliosus;

mechanical friction blistering;

corticosteroid atrophy; and

melanoma and non-melanoma skin cancer, including lignin melanoma, basalcell carcinoma, squamous cell carcinoma, actinic keratoses, and virallyinduced neoplasia (warts and condylomata accuminata).

Optimal methods and frequency of administration will be readily apparentto those skilled in the art or are capable of determination by routineexperimentation. Effective results in most cases are achieved by topicalapplication of a thin layer over the affected area, or the area whereone seeks to achieve the desired effect. Depending on the conditionbeing addressed, its stage or degree, and whether application is donefor therapeutic or preventive reasons, effective results are achievedwith application rates of from one application every two or three daysto four or more applications per day.

The invention is generally applicable to the treatment of the skin ofterrestrial mamnmals, including for example humans, domestic pets, andlivestock and other farm animals.

The following examples are offered for purposes of illustration, and arenot intended to limit nor to define the invention. All literaturecitations in these examples and throughout this specification areincorporated herein by references for all legal purposes to be servedthereby.

Materials and Methods for Examples 1 Through 11

A. Organ Culture Model and Measurement of Barrier Function

Timed pregnant Sprague-Dawley rats (plug date=day 0) were obtained fromSimonsen (Gilroy, Calif., USA) and fetuses were delivered prematurely onday 17. Transepidermal water loss (TEWL) was measured in excisedfull-thickness flank skin from the fetal rats after various times inculture. The skin explants were placed dermis-side down onto collagenmembrane inserts (3μ pore size) in medium M-199 (serum-free), andsubmerged, and the lateral edges and dermal surface were sealed withpetrolatum, such that water loss occurred only through the epidermalsurface. Explant samples were weighed hourly, at ambient temperature(24±3° C.) and humidity (40±5%), over 6 hours using a Cahn balance(sensitivity 0.001 mg). TEWL levels are reported as milligrams of waterlost per square millimeter of epidermal surface per hour.

The compounds used in these examples are as follows:

Prostaglandin J2 was obtained from Cayman Chemical Company (Ann Arbor,Mich., USA). Troglitazone was obtained from Parke-Davis Laboratories(Detroit, Mich., USA). The compound(4-chloro-6-(2,3-xylidino)-2-pyrimidinyl)thioacetic acid (known both aspirinixic acid and WY 14,643) were obtained from Wyeth Laboratories,Philadelphia, Pa., USA). The compounds cis-farnesol, nerolidol, andgeranylgeraniol were obtained from University of California, LosAngeles, Calif., USA. All other compounds were obtained from SigmaChemical Company (St. Louis, Mo., USA).

Fatty acids and clofibrate (p-chlorophenoxyisobutyric acid) were addedto the medium bound to 0.5% (weight/volume) bovine serum albumin (BSA).Isoprenoids and oxysterols were added in ethanol (≦0.1%) and juvenilehormone III was added as a dimethylsulfoxide (DMSO) solution (≦0.1%).Control explants were incubated in the presence of the appropriatevehicle (≦0.1% DMSO or ethanol, and/or ≦0.5% BSA).

B. Light and Electron Microscopy

Samples for light microscopy were fixed in modified Karnovsky'ssolution, plastic-embedded, and 0.5 μm sections were stained withtoluidine. Samples for electron microscopy were minced into 1 mm³pieces, fixed in modified Karnovsky's fixative, and processed. Sectionswere stained with uranyl acetate and lead citrate, post-fixed inruthenium tetroxide, and examined using a Zeiss 10 A electronmicroscope.

C. Tissue Preparation for Enzyme Assays

Epidermis was separated from dermis after incubation in 10 mMethylenediamine tetraacetic acid (EDTA) in Ca⁺⁺—and Mg⁺⁺—freephosphate-buffered saline (PBS), pH 7.4, at 37° C. for 30-40 minutes.The tissues were then minced, and homogenized on ice (three times at 15seconds each with a Polytron homogenizer, followed by sonication twiceat ten seconds each at 35% power) in either PBS containing 0.1 mMphenylmethylsulfonyl fluoride (PMSF) and 0.1% Triton X-100 (forβ-glucocerebrosidase) or in 10 mM Tris (pH 7.5) containing 0.15 Msucrose and 2 mM EDTA (for steroid sulfatase). β-Glucocerebrosidaseactivity was measured in the supernatant following centrifugation at10,000×g for fifteen minutes at 4° C. Steroid sulfatase activity wasmeasured in the microsomal fraction resulting from a 10,000×gcentrifugation (10 minutes, 4° C.) followed by 60 minutes of 100,000×gultracentrifugation at 4° C. Protein content was measured byconventional techniques.

D. β-Glucocerebrosidase and Steroid Sulfatase Activities

β-Glucocerebrosidase activity was assayed using the synthetic substrate4-methyl-umbelliferyl β-D-glucoside (4-MUG). The assays were performedin 5 mM sodium taurocholate in citrate-phosphate buffer (pH 5.6) with0.5 mM 4-MUG for sixty minutes at 37° C., with a final assay volume of100 μL, and protein concentration of 1-2 mg/mL. The reaction wasterminated with 2 mL of carbonate-bicarbonate buffer (pH 10.5). Thefluorescence was then measured at 360 λ (excitation) and 450 λ(emission) and compared with a standard 4-methylumbelliferone (4-MU)curve.

Steroid sulfatase activity was measured by incubating 100 μg ofmicrosomal protein in 0.1 M Tris containing 5.6 mM glucose (pH 7.4) with15 μM (³H) dihydroepiandrosterone sulfate (DHEAS) (15 μCi) in a finalvolume of 1.1 mL. The product, (³H) DHEA, was extracted with benzene andan aliquot counted by scintillation spectrophotometry.

E. Statistical Analysis

Statistical evaluation was performed using a Student's test.

EXAMPLE 1

The experiments reported in this example demonstrate that not allactivators of RXR heterodimers accelerate barrier development.Activators of three particular receptors—the retinoid receptor, thevitamin D receptor, and the peroxisome proliferator-activated receptor λ(PPARλ)—were used, and the negative results are demonstrated.

Prior studies have shown that full-thickness skin from gestational day17 rats, after two days in culture, exhibits lamellar bodies in thegranular cells and lamellar material secreted in the stratum corneuminterstices, but, like day 19 rat skin in utero, the epidermis lacksmature lameliar membrane structures and a competent barrier. Hanley, K.,et al., “Epidermal barrier ontogenesis: maturation in serum-free mediaand acceleration by glucocorticoids and thyroid hormone but not selectedgrowth factors,” J. Invest. Dermatol. 106:404-411 (1996). In contrast, astratum corneum with barrier function equivalent to that observed inmature epidermis normally forms by day 4 in culture, corresponding today 21 in utero. Hanley et al. (1996).

Skin explants from gestational day 17 rats were incubated in thepresence of either various activators or vehicle, and transepidermalwater loss was measured after two days. The activators and theconcentrations at which they were used were as follows:

9-cis-retinoic acid: an activator of the retinoid X receptor (RXR);concentration 1 μM

all-trans-retinoic acid: an activator of the retinoic acid receptor(RAR); concentration 1 μM

1,25-dihydroxyvitamin D3: an activator of the vitamin D receptor;concentration 1 μM

prostaglandin J2: an activator of PPARγ; concentration 10 μM

The results in terms of trans-epidernal water loss (TEWL) are shown inbar-graph form as the top four bars in FIG. 1, which when compared withthe control (the bottom bar in the Figure) indicate that none of thesefour activators had any significant effect on reducing the TEWL andhence promoting the barrier development.

In further tests, whose results are not shown in FIG. 1, the first threeactivators were tested at concentrations ranging from 1 nM to 1 μM, fortheir effect on the rate of barrier development. No effect was seen atany concentrations tested within this range.

These data demonstrate that activators of the retinoid receptors,vitamin D receptor and PPARγ do not accelerate fetal barrierdevelopment.

EXAMPLE 2

The experiments reported in this example demonstrate that activators ofPPARα accelerate barrier development, while activators of other PPARsubtypes do not.

Linoleic acid is an example of an activator of PPARα, and the TEWL valuefor explants incubated in 300 μM linoleic acid for two days is shown inFIG. 1. In contrast to the four ineffective activators discussed inExample 1 and the control, all of which are also shown in FIG. 1,linoleic acid markedly decreased the TEWL (p<0.005, n=8).

The three PPAR subtypes presently known are PPARα, PPARδ, and PPARγ.These subtypes are pharmacologically distinct and differentiallyactivated by various agents. Yu, V. C., et al., “RXRβ: a coregulatorthat enhances binding of retinoic acid, thyroid hormone, and vitamin Dreceptors to their cognate response elements,” Cell 67:1251-1266 (1991).To determine whether barrier development acceleration is attributablespecifically to PPARα, a variety of PPAR activators were tested, asfollows:

oleic acid: an activator for PPARα and suspected of also being anactivator for PPARδ, tested at a concentration of 300 μM

ETYA: an activator for PPARα and suspected of also being an activatorfor PPARδ, tested at a concentration of 100 μM

WY 14,643: an activator for PPARα only, tested at a concentration of 100μM

clofibrate: an activator for PPARα only, tested at a concentration of300 μM

troglitazone: an activator for PPARγ only, tested at a concentration of10 μM

Also relevant to this study is the test result for prostaglandin J2, anactivator for PPARγ only, which is shown in FIG. 1.

The results of the five activators listed above are shown in bar-graphform in FIG. 2, where two controls are included. All activators ofPPARα, including those known to activate only PPARα demonstrated asignificant positive effect, whereas the PPARγ activator troglitazone(like the PPARγ activator prostaglandin J2 shown in FIG. 1) had noeffect. These results indicate that activators of PPARα acceleratebarrier development while activators that activate only PPARγ do notaccelerate barrier development.

EXAMPLE 3

The experiments reported in this example demonstrate that activators offarnesol X-activated receptor (FXR) accelerate barrier development,while other compounds that are either similar in structure to farnesol,metabolites of farnesol, metabolic precursors of farnesol, or othermetabolites of metabolic precursors of farnesol, do not acceleratebarrier development.

Referring again to FIG. 1, farnesol is included in the bar graph, whichlists the TEWL value for explants incubated in 50 μM farnesol for twodays. In contrast to the four ineffective activators discussed inExample 1 and the control, farnesol markedly decreased the TEWL(p<0.005, n=8) to a similar degree as did linoleic acid.

To determine whether the effect on barrier development by farnesol ismediated by FXR, several other compounds related to farnesol in the waysstated or by similarly being known to activated FXR were tested forTEWL. Farnesol is produced by a multi-step metabolic synthesis fromacetyl coenzyme A, and one of the key intermediates is mevalonate. Aspart of the pathway, mevalonate is converted in a rate-limiting step toisopentenyl pyrophosphate which through a series of reactions convertsto farnesyl pyrophosphate. The latter is converted directly to farnesolbut is also capable of following separate pathways toward the synthesisof compounds such as cholesterol, ubiquinone, dolichol, carotenoids,vitamin D, bile acids and steroid hormones. The farnesol pathway in turnleads to farnesoid metabolites such as farnesal, farnesoic acid, methylfarnesoate, and juvenile hormone III.

This series of experiments therefore included tests on mevalonate(tested at 200 μM), juvenile hormone III (at 100 μM) and 25-hydroxycholesterol (at 50 μM), as well as nerolidol (at 100 μM),geranylgeraniol (at 50 μM), cis-farnesol (at 50 μM), and squalene (at 50μM), due the similarities in structure between the latter four compoundsand farnesol.

The results of these tests are shown in the bar graph of FIG. 3, whichindicates that juvenile hormone III at 100 μM significantly acceleratedbarrier formation (p<0.005, n=6). By contrast, neither mevalonate at 200μM, 25-hydroxy cholesterol at 50 μM, squalene at 50 μM, geranylgeraniolat 50 μM, cis-farnesol at 50 μM, nor nerolidol at 100 μM significantlyaffected barrier function. These results indicate that the accelerationof barrier development by farnesol and juvenile hormone III is FXRmediated.

EXAMPLE 4

The experiments reported in this example explore the dose responses ofthe various PPARα and FXR activators tested in the preceding examples.Tests for TEWL were conducted as described above, using clofibrate,oleic acid, linoleic acid, farnesol and juvenile hormone III, each overa range of concentrations. The results are plotted in FIGS. 4a(clofibrate and oleic acid), 4 b (linoleic acid) and 4 c (farnesol andjuvenile hormone III). For both clofibrate and oleic acid, maximaleffects on barrier development occur at a concentration of approximately500 μM, and half-maximal effects occur at approximately 250 μM. Forlinoleic acid, the maximal effect occurs at approximately 30 μM and thehalf-maximal effect at approximately 12.5 μM. Farnesol demonstrated amaximal effect at approximately 50 μM and a half-maximal effect atapproximately 20 μM. Juvenile hormone III demonstrated a maximal effectat approximately 250 μM and a half-maximal effect at approximately 75μM.

EXAMPLE 5

A series of experiments was performed to determine whether activation ofboth RXR and FXR, or both RXR and PPARα would result in barrierdevelopment to a greater degree than activation of FXR or PPARα alone,either in a synergistic or additive manner.

Explants were incubated in 9-cis-retinoic acid in combination first withclofibrate, then with farnesol, then with both, followed by measurementsof TEWL. The 9-cis-retinoic acid was tested at a concentration of 1 μM,while the clofibrate and farnesol were both tested at suboptimalconcentrations as determined in Example 4 above—i.e., clofibrate at 100μM and farnesol at 10 μM. No effect on either function or epidermalmorphologic maturation was observed by either combination.

Experiments were then performed to determine whether the combination ofa PPARα activator and an FXR activator would produce a synergisticresult or an additive result. A first set of experiments was performedusing suboptimal concentrations of these two activators (clofibrate at100 μM and farnesol at 10 μM). The results are plotted in the bar graphof FIG. 5a, where they are compared with a control (no activatorspresent). The TEWL value for clofibrate, represented by the bar labeled“Clo,” is identical to the data point in FIG. 4a at the sameconcentration, while the TEWL value for farnesol, represented in FIG. 5aby the bar labeled “Farn,” is identical to the data point in FIG. 4c atthe same concentration. The TEWL value for the combination of the twoactivators is represented in FIG. 5a by the bar labeled “Clo+Farn,” andshows that barrier development was significantly accelerated, indicatingan additive effect of clofibrate and farnesol.

In a second set of experiments, whose results are shown in FIG. 5b,farnesol and clofibrate were used at their maximal concentrations, asdetermined in FIGS. 4a and 4 c. The TEWL value for clofibrate in FIG. 5bis represented by the bar labeled “Clo” and is identical to the value inFIG. 4a at the maximally effective concentration, while the TEWL valuefor farnesol is represented in FIG. 5b by the bar labeled “Farn” and isidentical to the value in FIG. 4c at the maximally effectiveconcentration. The TEWL value for the combination of the two activatorsat their maximally effective concentrations is represented in FIG. 5b bythe bar labeled “Clo+Farn,” and shows that barrier development was notfurther accelerated as compared to the values for the two activatorsalone.

The additive effects of clofibrate and farnesol at suboptimal dosessuggest a similar activation pathway toward barrier development. This isfurther suggested by the lack of synergy in combining the same twoactivators at maximally effective concentrations. The lack of synergybetween 9-cis-retinoic acid and either clofibrate or farnesol suggestseither that the pathway toward barrier development is independent of RXRactivation, or that sufficient endogenous RXR activators were present.

EXAMPLE 6

This example seeks to determine whether a relationship exists at thelight microscope and electron microscope levels between the effects ofPPARα and FXR activators and epidermal maturation.

Explants incubated in various media were examined by light microscopy.The incubation media included control medium (containing no activators)and various media individually containing 300 μM clofibrate, 100 μMjuvenile hormone III, 300 μM oleic acid, 30 μM linoleic acid, 50 μMfarnesol, and various concentrations of 9-cis-retinoic acid,all-trans-retinoic acid, and 1,25-dihydroxy vitamin D3.

The light microscopy of the epidermis in the control explants showedthat these explants lacked both a distinct stratum granulosum and adistinct stratum corneum. In contrast, the explants incubated for 48hours in the clofibrate-containing medium, the juvenile hormoneIII-containing medium, the oleic acid-containing medium, the linoleicacid-containing medium, and the farnesol-containing medium all had botha multi-layered stratum granulosum and a stratum corneum. The explantsincubated in the retinoic acid-containing media (both 9-cis andall-trans) and those incubated in the 1,25-dihydroxy vitaminD3-containing medium did not exhibit a distinct stratum corneum, andwere morphologically indistinguishable from the controls.

Observations were then made of the ultrastructural maturation of theouter epidermis in explants that had been incubated in the various mediaand then post-fixed in ruthenium tetroxide. Media identical to those ofthe preceding paragraph were used, excluding the retinoicacid-containing and 1,25-dihydroxy vitamin D3-containing media. Explantstreated with all media containing FXR and PPARα activators showedmultiple arrays of mature lamellar membrane unit structures filling theextracellular domains of the stratum corneum. In neither the controls,the vitamin D3, the all-trans-retinoic acid, nor the 9-cis-retinoic acidcultures, were the extracellular lamellae in the single-layered stratumcorneum organized into mature lamellar membrane unit structures.

These results indicate that stimulation of the functional development ofthe barrier is accompanied by accelerated epidermal stratification anddifferentiation and the more rapid appearance of mature lamellar unitstructures in the stratum corneum.

EXAMPLE 7

This example investigates the effect of PPARα and FXR activators on theexpression of certain enzymes whose activities increases during barrierformation. It is known in the art that epidermal β-glucocerebrosidaseactivity increases during stratum corneum and barrier development in therat in utero and in vitro, that inhibition of this enzyme preventsnormal barrier formation, and that this enzyme is required for barrierhomeostasis (in vivo barrier function in adult). It has also been shownthat hormones that accelerate epidermal barrier formation increaseβ-glucocerebrosidase activity in fetal skin explants. Steroid sulfataseactivity is also known to increase during barrier formation, and is alsostimulated by hormones that accelerate barrier formation.β-Glucocerebrosidase and steroid sulfatase were thus selected as enzymesfor this study.

Skin explants from 17-day fetal rats were incubated for 24 or 48 hoursin media individually containing clofibrate (300 μM) and juvenilehormone III (100 μM), and control media. Enzyme activity was measuredand the results are presented in Table I as the mean values of fivedeterminations±SEM (standard error of the mean). All p values are ≦0.005compared with controls of the same time period.

TABLE I Enzyme Activity Activity β-Glucocerebrosidase Steroid Sulfatase(nmol/min/mg) (pmol/h/mg) 24 - Hour Incubation: Vehicle 1.35 ± 0.22 5.99± 0.80 Clofibrate 2.97 ± 0.30 13.90 ± 1.10  Juvenile 2.89 ± 0.20 12.55 ±1.25  Hormone III 48 - Hour Incubation: Vehicle 3.02 ± 0.45 8.75 ± 0.91Clofibrate 5.35 ± 0.89 22.51 ± 2.60  Juvenile 5.20 ± 0.62 18.23 ± 2.35 Hormone III

The data in this table indicate that β-glucocerebrosidase activity wasapproximately two-fold higher in treated explants than in controls afterboth 24 hours and 48 hours, and that steroid sulfatase activity was alsoincreased 1.6 times over controls after 24 hours, and 2.5 times after 48hours. These data demonstrate that both clofibrate and juvenile hormoneIII accelerate the developmental increases in activity of two lipidmetabolic enzymes associated with the formation of a competent barrier.

EXAMPLE 8

This example illustrates that activators of PPARα induce differentiationin keratinocytes, the differentiation being part of the process ofdeveloping a mature epidermal barrier. Involucrin and transglutaminase(and their mRNA) were measured as indicators of differentiation, thesetwo proteins being components of the ectoskeleton of the corneosites ofcells of the stratum corneum. The PPARα activator tested was clofibrate.

Human keratinocyte cells were incubated in culture containing calcium ata concentration of 0.03 mM Ca⁺⁺ (a level that is too low to inducedifferentiation). Also included in the culture medium were clofibrate at400 μM or ETYA at 10 μM and 20 μM, while separate cell cultures weremaintained as controls with neither clofibrate nor ETYA. The productionof mRNA for both involucrin and transglutaminase was measured atintervals over a 48-hour time period (0, 6, 12, 24, and 48 hours) byNorthern Blot analysis. The results for involucrin are shown in FIG. 6aand those for transglutaminase are shown in FIG. 6b. In each case, thelighter bars represent the control cells and the darker bars the cellsincubated in the clofibrate-containing medium. The degree of mRNAgenerated is expressed as a percent of the control at zero hours. Thedata in the figures indicates that clofibrate-treated cells exhibitsignificantly increased levels of both involucrin beginning at six hoursand transglutaminase beginning at twelve hours. Similar results wereobtained with ETYA.

The experiments were then repeated, except with the calcium at aconcentration of 1.2 mM Ca⁺⁺ in the media, a concentration high enoughto induce differentiation by itself. The results for involucrin areshown in FIG. 7a and those for transglutaminase are shown in FIG. 7b. Inthese figures as well, the lighter bars represent the control cells andthe darker bars the cells incubated in the clofibrate-containing medium,and the degree of mRNA generated is expressed as a percent of thecontrol at zero hours. In the control cells, the involucrin mRNA levelincreases to a maximum at 24 hours, then declines by 48 hours, while thetransglutaminase mRNA rises for the first 24 hours, then either levelsoff or continues to increase at a modest rate. The clofibrate-treatedcells exhibit increased mRNA levels of both involucrin andtransglutaminase at all time points relative to the controls. Similarresults were obtained with ETYA.

The dose dependency of clofibrate on the levels of the two mRNAs wasdetermined by a series of incubations for 24 hours using mediacontaining varying concentrations of clofibrate ranging from 0 (vehicleonly) to 400 μM, in either 0.03 mM or 1.2 mM Ca⁺⁺. The results in termsof both involucrin and transglutaminase mRNA levels are shown in FIG. 8,where the degree of mRNA generated is expressed as a percent of therespective measurements taken at zero hours. The mRNA levels for eachprotein are shown to increase with increasing dosages within this range.

Measurements of the level of the involucrin protein itself wereperformed in the culture medium by Western Blot. Various incubationswere performed, in media containing clofibrate at concentrations rangingfrom 0 (vehicle only) to 400 μM, and calcium ion at both 0.03 mM and 1.2mM. The results are shown in FIG. 9a for tests with calcium ion at 0.03mM, and in FIG. 9b for tests with calcium ion at 1.2 mM. In thesefigures, the protein contents are expressed as percents of therespective measurements taken from media incubated for the same periodof time but in the absence of clofibrate. At both calcium levels,substantial increases in the protein content were observed as theclofibrate concentration was increased within this range. Together,these data demonstrate that PPARα activators have a profound effect onthe expression of protein markers of epidermal differentiation.

EXAMPLE 9

This example demonstrates the increase in keratinocyte differentiationby FXR activators. This is an indication of increased expression of keyprotein markers of epidermal differentiation. The activators used werefarnesol and juvenile hormone III.

The procedures of Example 8 were followed, and measurements were takenon the involucrin protein levels from cells incubated at variousconcentrations of farnesol and juvenile hormone III ranging from 0(vehicle only) to 15 μM, in media containing 0.03 mM and 1.2 mM calciumion, for 24 hours. The results are shown in FIG. 10 for farnesol andFIG. 11 for juvenile hormone III, in 0.03 mM Ca⁺⁺. The figures showsubstantial increases in involucrin protein content as the farnesolconcentration was increased within this range, and the same was true forjuvenile hormone III. These data show that FXR activators increase theexpression of key protein markers of epidermal differentiation.

EXAMPLE 10

This example illustrates the effect of clofibrate, farnesol and juvenilehormone III on the rate of cornified envelope formation, as measured by³⁵S incorporation into detergent- and reducing agent-insoluble protein.

Normal human keratinocytes were cultured to 80% confluence in KGMcontaining 0.07 μM Ca⁺⁺. At 80% confluence, the cells were switched toKGM containing 0.03 μM Ca⁺⁺ or 1.2 μM Ca⁺⁺, plus varying concentrationsof clofibrate ranging from 0 to 400 μM. Cells were incubated in thesesolutions plus ³⁵S-methionine/cysteine for 48 hours. Ionomycin, 5 μM,was added at 46 hours, two hours before assaying for cornifiedenvelopes. Cells were rinsed with phosphate-buffered saline (PBS) andsolubilized in 2% sodium dodecylsulfate, and an aliquot was placed in 4%sodium dodecylsulfate/40 mM dithiothreitol in boiling water for 30minutes. The sodium dodecylsulfate-insoluble pellet was washed with 0.1%sodium dodecylsulfate/0.1% dithiothreitol, and the radioactivityincorporated into the detergent-insoluble cornified envelope wasdetermined by scintillation counting. To determine total proteinsynthesized during the ³⁵S-labeling period, an aliquot of cell lysatebefore boiling was precipitated with 10% trichloroacetic acid on ice forthirty minutes, washed with 5% trichloroacetic acid, and quantitated byscintillation counting. Percentage of cornified envelope was calculatedas percentage cpm/total protein cpm×100.

The results are shown in Table II, where they are expressed as percentof the value representing the control with 0.03 mM Ca⁺⁺, and where eachentry represents the mean±SEM of two independent experiments. For thedata at 0.03 mM Ca⁺⁺ and 200 and 400 μM clifobrate, and for the data at1.2 mM Ca⁺⁺ and 400 μM, the value of p was<0.01 relative to thecorresponding vehicle-only controls.

TABLE II Cornified Envelope Formation Induced by Clofibrate CornifiedEnvelope Calcium Content Clofibrate Content Formation in Medium inMedium (% of Control) 0.03 mM 0 100.0% ± 13.9 0.03 mM  50 μM 121.4% ±15.7 0.03 mM 200 μM 321.3% ± 30.4 0.03 mM 400 μM 386.8% ± 28.7 1.2 mM 0488.0% ± 34.1 1.2 mM  50 μM 458.9% ± 39.8 1.2 mM 200 μM 529.3% ± 17.41.2 mM 400 μM 581.9% ± 28.7

The data in Table II indicate that clofibrate increased cornifiedenvelope formation in both high and low Ca⁺⁺ media.

Similar tests were performed using farnesol at 10 μM and juvenilehormone III at 15 μM, individually in separate media at low calciumconcentration (0.03 mM). The 48-hour results are shown in FIG. 12, whichindicates that both farnesol and juvenile hormone III producedsubstantial increases in the rate of cornified envelope formation.

EXAMPLE 11

This example illustrates the effect of clofibrate, farnesol and juvenilehormone II on keratinocyte cell growth, demonstrating that both PPARαand FXR activators inhibit cell growth and proliferation.

Preconfluent keratinocytes were treated for 48 hours with varyingconcentrations of clofibrate ranging from 0 (vehicle only) to 400 μM, inboth low (0.03 mM) calcium and high (1.2 mM) calcium. The treatedkeratinocytes were harvested and sonicated, and the resultinghomogenates were incubated with 1 μL/mL bis-benzimidazole for two hoursin the dark. DNA content was quantified by reading the samples on aspectrofluorimeter. The results are shown in Table III. Each value inthe table is a mean of three samples.

TABLE III Effect of Clofibrate on Keratinocyte Growth DNA ContentClofibrate After 48 Hours (μM) (μg/dish) Significance  0 15.5 ± 0.9 — 50 14.9 ± 1.5 not significant 200 12.9 ± 1.1 p < 0.1 400 11.0 ± 1.3  p< 0.01

Similar tests were performed using farnesol at 10 μM and juvenilehormone III at 15 μM, both in the presence of 0.03 mM Ca⁺⁺ and with 48hours of incubation. The results are shown in FIG. 13. Table II and FIG.13 collectively show that both PPARα and FXR activators inhibit cellgrowth.

Examples 12 through 17 address the activity of oxysterol activators ofLXRα in the utilities addressed by this invention.

Materials and Methods for Examples 12 Through 17

A. Cell Culture

Human epidermis was isolated from newborn foreskins and keratinocyteswere plated in serum-free keratinocyte growth medium (“KGM”; Clonetics,San Diego, Calif. USA), using conventional techniques. Cells weretreated with either the test compounds in vehicle or vehicle alone(<0.1% ethanol) for 24 or 48 hours. The test compounds were obtainedfrom Sigma Chemical Co. (St. Louis, Mo. USA) and were stored as 15 mMstock solutions at −20° C. Mevalonate was solubilized in sterile water.

B. RNA Isolation, Northern Blotting and cDNA Probes

Total RNA was isolated by using TRIZOL (Sigma Chemical Co.), followingthe manufacturer's protocol. Ethanol-precipitate RNA pellets weresuspended in sterile, diethylpyrocarbonate (DEPC)-treated water, and RNAwas quantified by absorbance at 260 nm using the 260/280 run ratio as anindex of purity. RNA (15 μg per sample) was size fractionated through a1% agarose gel containing 2.2 M formaldehyde. RNA integrity wasvisualized following acridine orange staining of the gel followingelectrophoresis. The RNA was transferred to a nylon membrane that wassubsequently baked at 80° C. for 2 hours. Blots were hybridized with theappropriate ³²P-labeled probe overnight at 65° C. Washes were thenperformed in a solution containing 0.1% SSC and 0.1% sodium dodecylsulfate (SDS) for 20 minutes at room temperature, followed by a20-minute wash at 65° C. Autoradiography was performed at −70° C. Blotswere probed with β-actin to confirm equal loading. Appropriate bandswere quantified by densitometry.

C. Involucrin and Transglutaminase Protein Levels

Protein concentration was assessed by protein electrophoresis andWestern blotting. Cells were lysed in 2% SDS and the lysate sonicated.Protein determinations were made by use of a Bicinchoninic acid proteinassay (Pierce Chemical Company, Rockford, Ill. USA). Following proteindetermination, equal amounts of protein (50 μg) were separated byelectrophoresis on 7.5% polyacrylamide gets and electroblotted ontopolyvinylidene difluoride membranes (0.2μ, obtained from Bio-RadLaboratories, Hercules, Calif. USA). Involucrin protein was detected byincubation overnight at 4° C. with a polyclonal rabbit anti-humaninvolucrin antibody (1:1000 dilution). The involucrin-specific bands onthe autoradiograms were quantitated by densitometry. Transglutaminaseprotein expression was measured in a similar manner, using stacking,sample and running buffers containing 4 M urea, and the gels followingelectrophoresis were washed in 4 M urea, 25 mM TRIS—HCl, pH 7.4, 7.5 mMNaCl, 0.1 mM dithiothreitol (DTT), and 2 mM ethylenediamine tetraaceticacid for 90 minutes prior to electoblotting, allowing thetransglutaminase to be detected by antibody. Specific bands on theautoradiograms were quantified by densitometry.

D. Cornified Envelope Formation

To determine the rate of cornified envelope formation, cells werelabeled with 35S-methionine/cysteine (2 μCi/mL; trans 35S label, ICNBiomedical, Inc., Irvine, Calif. USA) for 48 hours, incubated for thelast 2 hours with 5 μM ionomycin, washed with phosphate-buffered saline(PBS), and harvested into 1.1 mL of 2% SDS. Aliquots were reserved forprotein determinations. The remaining cell lysate (1 mL) was sonicatedbriefly (10 seconds). One mL of 4% SDS/4 mM DTT was then added, and themixture was heated to >95° C. for 30 minutes. The mixture was thencooled and SDS/DTT—insoluble material was collected on filter discs,washed with 0.5% SDS/0.5% DTT, and quantitated by scintillationspectrophotometry. To determine total protein synthesized during the 48hours of ³⁵S labeling, a reserved aliquot of the cell lysate (takenprior to heating) was precipitated with an equal volume of 2% bovineserum albumin (BSA) and 1 mL of 10% (weight/volume) trichloroacetic acid(TCA) on ice for thirty minutes, and ³⁵S-labeled precipitated proteinwas collected onto filters (pore size P8, Fisher Scientific, Pittsburgh,Pa. USA), washed with 5% TCA, and quantified by scintillationspectroscopy. Total protein was determined by conventional methods.

E. DNA Synthesis

The rate of DNA synthesis was determined by measuring the incorporationof ³H-thymidine into cellular DNA after 16 hours of incubation with 2μCi ³H-thymidine per mL of media (110 Ci/mmol methyl-1′,2′-³H-thymidine(Amersham Laboratories, Arlington Heights, Ill. USA). The cells werethen solubilized in 1 N NaOH, and the radioactivity in the washed TCAprecipitate was quantitated by scintillation spectroscopy.

F. Transfections

Keratinocytes were transfected by passing primary keratinocytes onto6-well multiwell plates 1-2 days prior to transfection to yield aconfluence of 20-40% on the day of transfection. Involucrin promoterconstruct (1 μg), 0.1 μg RSV-β-gal, and 7.5 μg of polybrene(dihexabromide, Aldrich Chemical Company, inc., Milwaukee, Wis. USA)were added in media (KGM containing 0.03 mM Ca⁺⁺) in a final volume of0.35 mL, and keratinocytes were incubated at 37° C. for 5 hours withgentle shaking each hour. Cells were then rinsed with CMF PBS, followedby incubation at room temperature for three minutes with 10% glycerol inmedia. Following two rinses with CMF PBS, keratinocytes were incubatedovernight with 2 mL of KGM containing 0.03 mM Ca⁺⁺. Keratinocytes weretreated the following day with fresh media (either 0.03 or 1.2 mM Ca⁺⁺)containing either 10 μM of the test compound of vehicle (ethanol). Cellswere rinsed and harvested in 250 μL cell lysis buffer. The lysate wasspun at 10,000×g (4° C.) for two minutes, and 20 μL of supernatant wasassayed with luciferase substrate and β-galactosidase substrate.β-Galactosidase activity was used to normalize data and correct for anytransfection inefficiencies.

EXAMPLE 12

This example demonstrates that the oxysterol activators of LXRαstimulate keratinocyte differentiation, as indicated by the levels ofinvolucrin and transglutaminase mRNA, while cholesterol, mevalonate, and22(S)-hydroxycholesterol demonstrated no significant effects.

Northern blot analyses were performed on keratinocytes maintained in lowcalcium (0.03 mM) and incubated for 24 hours in the presence of vehiclealone (<0.1% ethanol) or vehicle containing 25-hydroxycholesterol (10μM), 22(R)-hydroxycholesterol (10 μM), cholesterol (10 μM), ormevalonate (500 μM) (individually). The mRNA levels of involucrin(“INV”) and transglutaminase (“TG'ase”) are shown in the bar graph ofFIG. 14a, the INV indicated by clear bars and the TG'ase by shaded bars,both with error limits shown. The figure shows that both25-hydroxycholesterol (“25-OH”) and 22(R)-hydroxy-cholesterol (“22R-OH”)exhibited an approximate twofold increase in mRNA levels compared to thevehicle alone. In contrast; neither cholesterol (“chol”) nor mevalonate(“mev”) had any effect.

As a further comparison, 22(R)-hydroxycholesterol and22(S)-hydroxycholesterol (“22S-OH”) were tested under the sameconditions, and the results are shown in the bar graph of FIG. 14b,using the same bar indications as those in FIG. 14a. No effect on eitherINV or TG'ase mRNA was observed in the 22S—OH data, while the 22R—OHdata repeated what was observed in FIG. 14a.

The dose dependency of 22(R)-hydroxycholesterol was then determined,using doses of 0, 0.1 μM, 1.0 μM, 10.0 μM, and 15.0 μM. The results areshown in the bar graph of FIG. 15, using the same bar indications forINV and TG'ase mRNA as in FIGS. 14a and 14 b. The plot indicates thatmaximal effects were seen with a dosage of 10-15 μM, and half-maximaleffects with approximately 5 μM.

EXAMPLE 13

An extracellular calcium concentration of 1.2 mM is well known tostimulate differentiation in keratinocytes. This example demonstratesthat oxysterol activators of LXRα in the presence of a high calciumconcentration stimulate keratinocyte differentiation even further.

The experiment of Example 12 was repeated except that the calciumconcentration was raised to 1.2 mM and only the vehicle alone and thevehicle plus 25-hydroxy-cholesterol (10 μM), 22(R)-hydroxycholesterol(10 μM), or cholesterol (10 μM) were tested. The results are shown inthe bar graph of FIG. 16, using the same bar indications as in thepreceding figures. The bar graph of FIG. 16 shows that25-hydroxycholesterol and 22(R)-hydroxycholesterol both induced mRNAlevels of both INV and TG'ase, INV by approximately 2.3-2.4 times thecontrol, and TG'ase by approximately 2.5-2.9 times the control, whilecholesterol had no effect on either. Determinations were also made ofβ-actin mRNA levels, and the results (not shown in the Figures)indicated that these levels were unaffected by oxysterol treatment ineither low (0.03 mM) or high (1.2 mM) calcium conditions.

EXAMPLE 14

This example demonstrates that the oxysterol activators of LXR stimulateprotein levels of involucrin and transglutaminase, while cholesteroldoes not.

Levels of involucrin and transglutaminase protein were measured inkeratinocytes incubated in low calcium (0.03 mM) and treated with thetest compounds for 24 hours. The results are shown in the bar graph ofFIG. 17a, where the test compounds are compared with the vehicle alone(control) and cholesterol, using the same bar indications as in FIGS. 14through 16. The plot shows that 25-hydroxycholesterol induced INV levelsapproximately 1.7-fold and TG'ase protein levels approximately 1.6-foldrelative to the control, and that 22(R)-hydoxycholesterol induced INVlevels approximately 3.2-fold and TG'ase protein levels approximately2.8-fold relative to the control. Cholesterol had no effect.

Tests performed in high calcium conditions (1.2 mM) are shown in FIGS.17b (for 22(R)-hydroxycholesterol only, at three dose levels) and 17 c(for 22(R)-hydroxycholesterol, 25-hydroxycholesterol, and cholesterol,all at a dose of 15 μM). At a concentration as low as 5 μM,22(R)-hydroxycholesterol showed a 2.1-fold increase in TG'ase proteinlevel (FIG. 17b). For INV protein, the increase at 15 μM concentrationwas 2.8-fold for 22(R)-hydroxycholesterol and 3.2-fold for25-hydroxycholesterol, with essentially no increase for cholesterol(FIG. 17c).

EXAMPLE 15

An additional measure of keratinocyte differentiation is the rate ofcornified envelope formation. Using the procedures set forth in theMaterials and Methods section above, 25-hydroxycholesterol (10 μM) and22(R)-hydroxycholesterol (10 μM) were compared with cholesterol (10 μM)and the vehicle alone (control), with treatment for 48 hours under bothlow calcium (0.03 mM) and high calcium (1.2 mM) conditions. The resultsare shown in the bar graphs of FIGS. 18a (low calcium) and 18 b (highcalcium). The low calcium results (FIG. 18a) showed that treatment with25-hydroxycholesterol yielded a 1.6-fold increase in cornified envelope(CE) formation, and treatment with 22(R)-hydroxycholesterol yielded a1.3-fold increase. The high calcium results (FIG. 18b) showed thattreatment with 25-hydroxycholesterol yielded a 1.75-fold increase in CEformation, and treatment with 22(R)-hydroxycholesterol yielded a2.1-fold increase. Cholesterol itself produced no increase in either lowor high calcium conditions.

EXAMPLE 16

This example demonstrates that oxysterol activators of LXRα inhibitproliferation of keratinocytes, as indicated by the rate of DNAsynthesis in keratinocytes. Oxysterols are known to be potent inhibitorsof cell growth in other cell types such as thymocytes and lymphocytes.

Using the procedures set forth in the Materials and Methods sectionabove, 25-hydroxycholesterol (10 μM) and 22(R)-hydroxycholesterol (10μM) were compared with cholesterol (10 μM) and the vehicle alone(control), with treatment for 24 hours under low calcium (0.03 mM)condition. The results are shown in the bar graph of FIG. 19. The bargraph shows that 25-hydroxycholesterol decreased the rate of DNAsynthesis to 50% of the control, and 22(R)-hydroxycholesterol reducedthe rate to 42% of control, during the 16-hour time period in which theDNA synthesis was measured. In contrast, cholesterol modestly butsignificantly increased DNA synthesis (117% of control).

EXAMPLE 17

This example explores whether the increase that oxysterols cause in INVand TG'ase mRNA levels is related to the fact that these oxysterolsinhibit the enzyme HMG CoA reductase, which is the rate-limiting enzymeof cholesterol synthesis and which leads to decreased levels ofisoprenoids. To anwer this question, tests similar to those described inExample 12 above were performed in the presence and absence ofmevalonate, which is the earliest product of HMG CoA reductase.

The tests measured INV and TG'ase mRNA levels in keratinocytes treatedwith 25-hydroxycholesterol (10 μM), alone or in the presence ofmevalonate (10 μM), as well as mevalonate alone (10 μM), for 24 hours.Treatment with mevalonate alone had no effect on the INV and TG'ase mRNAlevels. Treatment with 25-hydroxycholesterol, both alone and in thepresence of mevalonate, resulted in a two-fold increase in the INV andTG'ase mRNA levels. The conclusion is that inhibition of HMG CoAreductase is not the basis for the increase in INV and TG'ase mRNAlevels caused by oxysterols.

The foregoing is offered primarily for purposes of illustration. It willbe readily apparent to those skilled in the art that the concentrations,operating conditions, materials, procedural steps and other parametersof protocols described herein may be further modified or substituted invarious ways without departing from the spirit and scope of theinvention.

We claim:
 1. A method for treating the epidermis of a terrestrialmammalian subject suffering from a perturbed epidermal barrier function,said method comprising topically administering to said epidermis atopical composition comprising an active ingredient that is an oxysterolactivator of LXRα selected from the group consisting of22(R)-hydroxycholesterol, 25-hydroxycholesterol, 7a-hydroxycholesterol,24-hydroxycholesterol, 27-hydroxycholesterol, 40-hydroxycholesterol,20,22-dihydroxycholesterol, and 20(S)-hydroxycholesterol, said activeingredient being present in a concentration that is effective inenhancing barrier development.
 2. A method in accordance with claim 1 inwhich said activator of LXRα is 22(R)-hydroxycholesterol.
 3. A method inaccordance with claim 1 in which said activator of LXRα is25-hydroxycholesterol.
 4. A method in accordance with claim 1 in whichthe concentration of said active ingredient is from about 10 μM to about1000 μM.
 5. A method for treating the epidermis or mucous membrane of aterrestrial mammalian subject suffering from a condition of disturbeddifferentiation or excess proliferation, said method comprisingtopically administering to said epidermis or mucous membrane a topicalcomposition comprising an active ingredient that is an oxysterolactivator of LXRα selected from the group consisting of22(R)-hydroxycholesterol, 25-hydroxycholesterol, 7a-hydroxycholesterol,24-hydroxycholesterol, 27-hydroxycholesterol, 4β-hydroxycholesterol,20,22-dihydroxycholesterol, and 20(S)-hydroxycholesterol, said activeingredient being present in a concentration that is effective innormalizing said condition.
 6. A method in accordance with claim 5 inwhich said activator of LXRα is 22(R)-hydroxycholesterol.
 7. A method inaccordance with claim 5 in which the concentration of said activeingredient is from about 10 μM to about 1000 μM.